Peptides and compounds that bind to a receptor

ABSTRACT

Peptides and compounds that bind to and activate the thrombopoietin receptor (c-mpl or TPO-R) or otherwise act as a TPO agonist are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application which is a continuation-in-part of U.S. applicationSer. No. 10/918,561, filed on Aug. 13, 2004, claims priority to U.S.application Ser. No. 10/918,561 and 60/498,740, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides peptides and compounds that bind to andactivate the thrombopoietin receptor (c-mpl or TPO-R) or otherwise actas a TPO agonist. The invention has application in the fields ofbiochemistry and medicinal chemistry and particularly provides TPOagonists for use in the treatment of human disease.

BACKGROUND OF THE INVENTION

Megakaryocytes are bone marrow-derived cells, which are responsible forproducing circulating blood platelets. Although comprising <0.25% of thebone marrow cells in most species, they have >10 times the volume oftypical marrow cells. See Kuter, et. al., Proc. Natl. Acad. Sci. USA91:11104-11108 (1994). Megakaryocytes undergo a process known asendomitosis whereby they replicate their nuclei but fail to undergo celldivision and thereby give rise to polyploid cells. In response to adecreased platelet count, the endomitotic rate increases, higher ploidymegakaryocytes are formed, and the number of megakaryocytes may increaseup to 3-fold. See Harker, J. Clin. Invest., 47:458-465 (1968). Incontrast, in response to an elevated platelet count, the endomitoticrate decreases, lower ploidy megakaryocytes are formed, and the numberof megakaryocytes may decrease by 50%.

The exact physiological feedback mechanism by which the mass ofcirculating platelets regulates the endomitotic rate and number of bonemarrow megakaryocytes is not known. The circulating thrombopoieticfactor involved in mediating this feedback loop is now thought to bethrombopoietin (TPO). More specifically, TPO has been shown to be themain humoral regulator in situations involving thrombocytopenia. See,e.g., Metcalf, Nature, 369:519-520 (1994). TPO has been shown in severalstudies to increase platelet counts, increase platelet size, andincrease isotope incorporation into platelets of recipient animals.Specifically, TPO is thought to affect megakaryocytopoiesis in severalways: (1) it produces increases in megakaryocyte size and number; (2) itproduces an increase in DNA content, in the form of polyploidy, inmegakaryocytes; (3) it increases megakaryocyte endomitosis; (4) itproduces increased maturation of megakaryocytes; and (5) it produces anincrease in the percentage of precursor cells, in the form of smallacetylcholinesterase-positive cells, in the bone marrow.

Because platelets (thrombocytes) are necessary for blood clotting andwhen their numbers are very low a patient is at serious risk of deathfrom catastrophic hemorrhage, TPO has potential useful application inboth the diagnosis and the treatment of various hematological disorders,for example, diseases primarily due to platelet defects. Ongoingclinical trials with TPO have indicated that TPO can be administeredsafely to patients. In addition, recent studies have provided a basisfor the projection of efficacy of TPO therapy in the treatment ofthrombocytopenia, and particularly thrombocytopenia resulting fromchemotherapy, radiation therapy, or bone marrow transplantation astreatment for cancer or lymphoma. See, e.g., McDonald, Am. J. Ped.Hematology/Oncology, 14:8-21 (1992).

The gene encoding TPO has been cloned and characterized. See Kuter, etal., Proc. Natl. Acad. Sci. USA, 91:11104-11108 (1994); Barley, et al.,Cell 77:1117-1124 (1994); Kaushansky et al., Nature 369:568-571 (1994);Wendling, et al., Nature, 369:571-574 (1994); and Sauvage et al., Nature369:533-538 (1994). Thrombopoietin is a glycoprotein with at least twoforms, with apparent molecular masses of 25 kDa and 31 kDa, with acommon N-terminal amino acid sequence. See, Bartley, et al., Cell,77:1117-1124 (1994). Thrombopoietin appears to have two distinct regionsseparated by a potential Arg-Arg cleavage site. The amino-terminalregion is highly conserved in man and mouse, and has some homology witherythropoietin and interferon-a and interferon-b. The carboxy-terminalregion shows wide species divergence.

The DNA sequences and encoded peptide sequences for human TPO-R (alsoknown as c-mpl) have been described. See Vigon, et al., Proc. Natl.Acad. Sci. USA, 89:5640-5644 (1992). TPO-R is a member of thehematopoietin growth factor receptor family, a family characterized by acommon structural design of the extracellular domain, including fourconserved C residues in the N-terminal portion and a WSXWS motif (SEQ IDNO: 1) close to the transmembrane region. See Bazan, Proc. Natl. Acad.Sci. USA, 87:6934-6938 (1990). Evidence that this receptor plays afunctional role in hematopoiesis includes observations that itsexpression is restricted to spleen, bone marrow, or fetal liver in mice(see Souyri, et al., Cell 63:1137-1147 (1990)) and to megakaryocytes,platelets, and CD34+ cells in humans (see Methia, et al., Blood82:1395-1401 (1993)). Furthermore, exposure of CD34+ cells to syntheticoligonucleotides antisense to mpl RNA significantly inhibits theappearance of megakaryocyte colonies without affecting erythroid ormyeloid colony formation. Some workers postulate that the receptorfunctions as a homodimer, similar to the situation with the receptorsfor G-CSF and erythropoietin.

The availability of cloned genes for TPO-R facilitates the search foragonists of this important receptor. The availability of the recombinantreceptor protein allows the study of receptor-ligand interaction in avariety of random and semi-random peptide diversity generation systems.These systems include the “peptides on plasmids” system described inU.S. Pat. Nos. 5,270,170 and 5,338,665; the “peptides on phage” systemdescribed in U.S. patent application Ser. No. 07/718,577, filed Jun. 20,1991, U.S. patent application Ser. No. 07/541,108, filed Jun. 20, 1990,and in Cwirla, et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990);the “polysome” system described in U.S. patent application Ser. No.08/300,262, filed Sep. 2, 1994, which is a continuation-in-partapplication based on U.S. patent application Ser. No. 08/144,775, filedOct. 29, 1993 and PCT WO 95/11992; the “encoded synthetic library”system described in U.S. patent application Ser. No. 08/146,886, filedNov. 12, 1993, Ser. No. 07/946,239, filed Sep. 16, 1992, and Ser. No.07/762,522, filed Sep. 18, 1991; and the “very large scale immobilizedpolymer synthesis” system described in U.S. Pat. No. 5,143,854; PCTPatent Publication No. 90/15070, published Dec. 13, 1990; U.S. patentapplication Ser. No. 07/624,120, filed Dec. 6, 1990; Fodor, et al.,Science, 251:767-773 (2/1991); Dower, et al., Ann. Rep. Med. Chem.,26:271-180 (1991); and U.S. patent application Ser. No. 07/805,727,filed Dec. 6, 1991; each of the foregoing patent applications andpublications is incorporated herein by reference.

The slow recovery of platelet levels in patients suffering fromthrombocytopenia is a serious problem, and has lent urgency to thesearch for a blood growth factor agonist able to accelerate plateletregeneration. The present invention provides such an agonist.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the novel and unexpecteddiscovery that a defined low molecular weight peptide and peptidemimetic has strong binding properties to the TPO-R and can activate theTPO-R. Accordingly, the peptides and peptide mimetics can be useful fortherapeutic purposes in treating conditions mediated by TPO (e.g.,thrombocytopenia resulting from chemotherapy, radiation therapy, or bonemarrow transfusions) as well as for diagnostic purposes in studying themechanism of hematopoiesis and for the in vitro expansion ofmegakaroycytes and committed progenitor cells.

Peptides and peptide mimetics suitable for therapeutic and/or diagnosticpurposes have an IC₅₀ of about 2 mM or less, as determined by thebinding affinity assay set forth in Example 3 below wherein a lower IC₅₀correlates to a stronger binding affinity to TPO-R. For pharmaceuticalpurposes, the peptides and peptidomimetics (or peptidemimetics)preferably have an IC₅₀ of no more than about 100 μM, more preferably,no more than 500 nM. In a preferred embodiment, the molecular weight ofthe peptide or peptide mimetic is from about 250 to about 8,000 daltons.If the peptides of this invention are oligomerized, dimerized and/orderivatized with a hydrophilic polymer as described herein, themolecular weights of such peptides will be substantially greater and canrange anywhere from about 500 to about 120,000 daltons, more preferablefrom about 8,000 to about 80,000 daltons.

When used for diagnostic purposes, the peptides and peptide mimetics ofthe present invention preferably are labeled with a detectable labeland, accordingly, the peptides and peptide mimetics without such a labelserve as intermediates in the preparation of labeled peptides andpeptide mimetics.

A peptide meeting the defined criteria for molecular weight and bindingaffinity for TPO-R comprise 9 or more amino acids wherein the aminoacids are naturally occurring or synthetic (non-naturally occurring)amino acids.

Accordingly, preferred peptides and peptide mimetics comprise a compoundhaving:

-   (1) a molecular weight of less than about 5000 daltons, and-   (2) a binding affinity to TPO-R as expressed by an IC₅₀ of no more    than about 100 μM,    wherein from zero to all of the —C(O)NH— linkages of the peptide    have been replaced by a linkage selected from the group consisting    of a —CH₂OC(O)NR— linkage; a phosphonate linkage; a —CH₂S(O)₂NR—    linkage; a —CH₂NR— linkage; and a—C(O)NR⁶—linkage; and a —NHC(O)NH—    linkage where R is hydrogen or lower alkyl and R⁶ is lower alkyl,    further wherein the N-terminus of said peptide or peptide mimetic is    selected from the group consisting of a —NRR¹ group; a —NRC(O)R    group; a —NRC(O)OR group; a —NRS(O)₂R group; a —NHC(O)NHR group; a    succinimide group; a benzyloxycarbonyl-NH—group; and a    benzyloxycarbonyl-NH—group having from 1 to 3 substituents on the    phenyl ring selected from the group consisting of lower alkyl, lower    alkoxy, chloro, and bromo, where R and R¹ are independently selected    from the group consisting of hydrogen and lower alkyl,    and still further wherein the C-terminus of said peptide or peptide    mimetic has the formula —C(O)R² where R² is selected from the group    consisting of hydroxy, lower alkoxy, and —NR³R⁴ where R³ and R⁴ are    independently selected from the group consisting of hydrogen and    lower alkyl and where the nitrogen atom of the —NR³R4 group can    optionally be the amine group of the N-terminus of the peptide so as    to form a cyclic peptide,    and physiologically acceptable salts thereof.

In a related embodiment, the invention is directed to a labeled peptideor peptide mimetic comprising a peptide or peptide mimetic described asabove having covalently attached thereto a label capable of detection.

In one embodiment, the core peptide comprises a sequence of amino acids:(SEQ ID NO:2) X₉ X₈ G X₁ X₂ X₃ X₄ X₅ X₆ X₇where X₉ is A, C, E, G, I, L, M, P, R, Q, S, T, or V; and X₈ is A, C, D,E, K, L, Q, R, S, T, or V; and X₆ is a b-(2-naphthyl)alanine (referredto herein as “2-Nal”) residue. More preferably, X₉ is A or I; and X₈ isD, E, or K. Further X₁ is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R, S, Tor V; X₃ is C, F, I, L, M, R, S, V or W; X₄ is any of the 20 geneticallycoded L-amino acids; X₅ is A, D, E, G, K, M, Q, R, S, T, V or Y; and X₇is C, G, I, K, L, M, N, R or V.

A particularly preferred peptide includes the amino acid sequence (SEQID NO:3): I E G P T L R Q (2-Nal) L A A R A.

In another embodiment, the peptide compounds of the present inventionare preferably dimerized or oligomerized to increase the affinity and/oractivity of the compounds. An example of a preferred dimerized peptidecompound includes, but is not limited to, the following:

Where X₁₀ is a sarcosine or β-alanine residue (SEQ ID NO:4). The abovestructure can also be represented by the following structure:(H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH₂.

In yet a further embodiment, preferred peptides for use in thisinvention include peptides that are covalently attached to one or moreof a variety of hydrophilic polymers. Suitable hydrophilic polymersinclude, but are not limited to, polyalkylethers as exemplified bypolyethylene glycol and polypropylene glycol, polylactic acid,polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc., as described in U.S. Pat. No. 5,869,451, theentire content of which is hereby incorporated by reference.

The compounds described herein are useful for the prevention andtreatment of diseases mediated by TPO, and particularly for treatinghematological disorders, including but not limited to, thrombocytopeniaresulting from chemotherapy, radiation therapy, or bone marrowtransfusions. Thus, the present invention also provides a method fortreating wherein a patient having a disorder that is susceptible totreatment with a TPO agonist receives, or is administered, atherapeutically effective dose or amount of a compound of the presentinvention.

The invention also provides for pharmaceutical compositions comprisingone or more of the compounds described herein and a physiologicallyacceptable carrier. These pharmaceutical compositions can be in avariety of forms including oral dosage forms, as well as inhalablepowders and solutions and injectable and infusible solutions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows and compares the activity of different compounds.

FIG. 2 shows and compares the activity of different compounds.

FIG. 3 shows and compares the in vivo change in platelet counts in ratdemonstrating the relative potency of PEGylated compounds.

FIGS. 4 and 5 show and compare the number and volume of circulatingplatelets in a dose dependent manner, respectively.

FIG. 6 shows that single intravenous doses of TPO Compound No. 1 (30,100 or 300 μg/kg) result in an increased peripheral platelet count innormal male Wistar rats.

FIG. 7 shows the PK, concentration—time profiles of TPO Compound No. 1in healthy male volunteers: filled square—TPO Compound No. 1, 0.75 μg/kgi.v.; open diamond—TPO Compound No. 1, 1.5 μg/kg i.v.; filledupward-triangle—TPO Compound No. 1, 2.25 μg/kg i.v.; opendownward-triangle—TPO Compound No. 1, 3 μg/kg i.v.

FIG. 8 shows that the platelet counts increased dose-dependently inhealthy male volunteers post administration of TPO Compound No. 1.

FIG. 9 shows that endogenous TPO levels increased dose-dependently inhealthy male volunteers post administration of TPO Compound No. 1.

DESCRIPTION OF SPECIFIC EMBODIMENTS

I. Definitions And General Parameters

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

“Agonist” refers to a biologically active ligand which binds to itscomplementary biologically active receptor and activates the lattereither to cause a biological response in the receptor or to enhancepreexisting biological activity of the receptor.

“Pharmaceutically acceptable salts” refer to the non-toxic alkali metal,alkaline earth metal, and ammonium salts commonly used in thepharmaceutical industry including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium, and protamine zinc salts, whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts, which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate, and the like.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, menthanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. For a descriptionof pharmaceutically acceptable acid addition salts as prodrugs, seeBundgaard, H., supra.

“Pharmaceutically acceptable ester” refers to those esters which retain,upon hydrolysis of the ester bond, the biological effectiveness andproperties of the carboxylic acid or alcohol and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableesters as prodrugs, see Bundgaard, H., ed., Design of Prodrugs, ElsevierScience Publishers, Amsterdam (1985). These esters are typically formedfrom the corresponding carboxylic acid and an alcohol. Generally, esterformation can be accomplished via conventional synthetic techniques.(See, e.g., March, Advanced Organic Chemistry, 4th Ed., John Wiley &Sons, New York (1992), 393-396 and references cited therein, and Mark,et al., Encyclopedia of Chemical Technology, John Wiley & Sons, New York(1980).) The alcohol component of the ester will generally comprise (i)a C₂-C₁₂ aliphatic alcohol that can or can not contain one or moredouble bonds and can or can not contain branched carbons or (ii) aC₇-C₁₂ aromatic or heteroaromatic alcohols. This invention alsocontemplates the use of those compositions which are both esters asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

“Pharmaceutically acceptable amide” refers to those amides which retain,upon hydrolysis of the amide bond, the biological effectiveness andproperties of the carboxylic acid or amine and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableamides as prodrugs, see Bundgaard, H., ed., Design of Prodrugs, ElsevierScience Publishers, Amsterdam (1985). These amides are typically formedfrom the corresponding carboxylic acid and an amine. Generally, amideformation can be accomplished via conventional synthetic techniques.(See, e.g., March, Advanced Organic Chemistry, 4th Ed., John Wiley &Sons, New York (1992), p. 393 and Mark, et al. Encyclopedia of ChemicalTechnology, John Wiley & Sons, New York (1980).) This invention alsocontemplates the use of those compositions which are both amides asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

“Pharmaceutically or therapeutically acceptable carrier” refers to acarrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is not toxic tothe host or patient.

“Stereoisomer” refers to a chemical compound having the same molecularweight, chemical composition, and constitution as another, but with theatoms grouped differently. That is, certain identical chemical moietiesare at different orientations in space and, therefore, when pure, hasthe ability to rotate the plane of polarized light. However, some purestereoisomers may have an optical rotation that is so slight that it isundetectable with present instrumentation. The compounds of the instantinvention may have one or more asymmetrical carbon atoms and thereforeinclude various stereoisomers. All stereoisomers are included within thescope of the invention.

“Therapeutically- or pharmaceutically-effective amount” as applied tothe compositions of the instant invention refers to the amount ofcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. In thepresent invention, the result will typically involve a decrease in theimmunological and/or inflammatory responses to infection or tissueinjury.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. Additionally, t-Buo is tert-bulyloxy, Bzl isbenzyl, CHA is cyclohexylamine, Ac is acetyl, Me is methyl, Pen ispenicillamine, Aib is aminoisobutyric acid, Nva is norvaline, Abu isaminobutyric acid, Thi is thienylalanine, OBn is O-benzyl, and hyp ishydroxyproline.

In addition to peptides consisting only of naturally-occurring aminoacids, peptidomimetics or peptide analogs are also provided. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptidemimetics” or “peptidemimetics” or “peptidomimetics” (Luthman, et al., A Textbook of DrugDesign and Development, 14:386-406, 2nd Ed., Harwood Academic Publishers(1996); Joachim Grante, Angew. Chem. Int. Ed. Engl., 33:1699-1720(1994); Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and FreidingerTINS, p. 392 (1985); and Evans, et al., J. Med. Chem. 30:1229 (1987),which are incorporated herein by reference). Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent or enhanced therapeutic or prophylactic effect.Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as naturally-occurring receptor-bindingpolypeptide, but have one or more peptide linkages optionally replacedby an alternative linkage such as —CH₂NH—, —CH₂S—, etc. by methods knownin the art and further described in the following references: Spatola,A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, andProteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, PeptideBackbone Modifications (general review); Morley, Trends Pharm. Sci. pp.463-468 (1980), (general review); Hudson, et al., Int. J. Pept. Prot.Res., 14:177-185 (1979); Spatola, et al., Life Sci., 38:1243-1249(1986); Hann, J. Chem. Soc. Perkin Trans. 1,307-314 (1982); Almquist, etal., J. Med. Chem., 23:1392-1398, (1980); Jennings-White, et al.,Tetrahedron Lett. 23:2533 (1982); Szelke, et al., European Appln. EP45665 (1982); Holladay, et al., Tetrahedron Lett., 24:4401-4404 (1983);and Hruby, Life Sci., 31:189-199 (1982); each of which is incorporatedherein by reference. A particularly preferred non-peptide linkage is—CH₂NH—. Such peptide mimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others. Labeling of peptidomimetics usually involvescovalent attachment of one or more labels, directly or through a spacer(e.g., an amide group), to non-interfering position(s) on thepeptidomimetic that are predicted by quantitative structure-activitydata and/or molecular modeling. Such non-interfering positions generallyare positions that do not form direct contacts with themacromolecules(s) (e.g., immunoglobulin superfamily molecules) to whichthe peptidomimetic binds to produce the therapeutic effect.Derivitization (e.g., labeling) of peptidomimetics should notsubstantially interfere with the desired biological or pharmacologicalactivity of the peptidomimetic. Generally, peptidomimetics ofreceptor-binding peptides bind to the receptor with high affinity andpossess detectable biological activity (i.e., are agonistic orantagonistic to one or more receptor-mediated phenotypic changes).

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo, et al., Ann. Rev. Biochem., 61:387 (1992),incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

Synthetic or non-naturally occurring amino acids refer to amino acidswhich do not naturally occur in vivo but which, nevertheless, can beincorporated into the peptide structures described herein. Preferredsynthetic amino acids are the D-α-amino acids of naturally occurringL-α-amino acid as well as non-naturally occurring D- and L-α-amino acidsrepresented by the formula H₂NCHR⁵COOH where R⁵ is 1) a lower alkylgroup, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3) aheterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selectedfrom the group consisting of oxygen, sulfur, and nitrogen, 4) anaromatic residue of from 6 to 10 carbon atoms optionally having from 1to 3 substituents on the aromatic nucleus selected from the groupconsisting of hydroxyl, lower alkoxy, amino, and carboxyl, 5)-alkylene-Ywhere alkylene is an alkylene group of from 1 to 7 carbon atoms and Y isselected from the group consisting of (a) hydroxy, (b) amino, (c)cycloalkyl and cycloalkenyl of from 3 to 7 carbon atoms, (d) aryl offrom 6 to 10 carbon atoms optionally having from 1 to 3 substituents onthe aromatic nucleus selected from the group consisting of hydroxyl,lower alkoxy, amino and carboxyl, (e) heterocyclic of from 3 to 7 carbonatoms and 1 to 2 heteroatoms selected from the group consisting ofoxygen, sulfur, and nitrogen, (f) —C(O)R² where R² is selected from thegroup consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and—NR³R⁴ where R3 and R⁴ are independently selected from the groupconsisting of hydrogen and lower alkyl, (g)-S(O)_(n) R⁶ where n is aninteger from 1 to 2 and R⁶ is lower alkyl and with the proviso that R⁵does not define a side chain of a naturally occurring amino acid.

Other preferred synthetic amino acids include amino acids wherein theamino group is separated from the carboxyl group by more than one carbonatom such as alanine, gamma-aminobutyric acid, and the like.

Particularly preferred synthetic amino acids include the D-amino acidsof naturally occurring L-amino acids and in particularL-(2-naphthyl)-alanine (2-Nal).

“Detectable label” refers to materials, which when covalently attachedto the peptides and peptide mimetics of this invention, permit detectionof the peptide and peptide mimetics in vivo in the patient to whom thepeptide or peptide mimetic has been administered. Suitable detectablelabels are well known in the art and include, by way of example,radioisotopes, fluorescent labels (e.g., fluorescein), and the like. Theparticular detectable label employed is not critical and is selectedrelative to the amount of label to be employed as well as the toxicityof the label at the amount of label employed. Selection of the labelrelative to such factors is well within the skill of the art.

Covalent attachment of the detectable label to the peptide or peptidemimetic is accomplished by conventional methods well known in the art.For example, when the 125 I radioisotope is employed as the detectablelabel, covalent attachment of 125 I to the peptide or the peptidemimetic can be achieved by incorporating the amino acid tyrosine intothe peptide or peptide mimetic and then iodimating the peptide (see,e.g., Weaner, et al., Synthesis and Applications of IsotopicallyLabelled Compounds, pp. 137-140 (1994)). If tyrosine is not present inthe peptide or peptide mimetic, incorporation of tyrosine to the N or Cterminus of the peptide or peptide mimetic can be achieved by well knownchemistry. Likewise, 32 P can be incorporated onto the peptide orpeptide mimetic as a phosphate moiety through, for example, a hydroxylgroup on the peptide or peptide mimetic using conventional chemistry.

II. Overview

The present invention provides compounds that bind to and activate theTPO-R or otherwise behave as a TPO agonist. These compounds include“lead” peptide compounds and “derivative” compounds constructed so as tohave the same or similar molecular structure or shape as the leadcompounds but that differ from the lead compounds either with respect tosusceptibility to hydrolysis or proteolysis and/or with respect to otherbiological properties, such as increased affinity for the receptor. Thepresent invention also provides compositions comprising an effectiveamount of a TPO agonist, and more particularly a compound, that isuseful for treating hematological disorders, and particularly,thrombocytopenia associated with chemotherapy, radiation therapy, orbone marrow transfusions.

II. Identification Of TPO-Agonists

Peptides having a binding affinity to TPO-R can be readily identified byrandom peptide diversity generating systems coupled with an affinityenrichment process.

Specifically, random peptide diversity generating systems include the“peptides on plasmids” system described in U.S. Pat. Nos. 5,270,170 and5,338,665; the “peptides on phage” system described in U.S. patentapplication Ser. No. 07/718,577, filed Jun. 20, 1991 which is acontinuation in part application of U.S. patent application Ser. No.07/541,108, filed Jun. 20, 1990, and in Cwirla, et al., Proc. Natl.Acad. Sci. USA, 87:6378-6382 (1990); the “polysome system” described inU.S. patent application Ser. No. 08/300,262, filed Sep. 2, 1994, whichis a continuation-in-part application based on U.S. patent applicationSer. No. 08/144,775, filed Oct. 29, 1993 and PCT WO 95/11992; the“encoded synthetic library (ESL)” system described in U.S. patentapplication Ser. No. 08/146,886, filed Nov. 12, 1993 which is acontinuation in part application of U.S. patent application Ser. No.07/946,239, filed Sep. 16, 1992, which is a continuation in partapplication of U.S. patent application Ser. No. 07/762,522, filed Sep.18, 1991; and the “very large scale immobilized polymer synthesis”system described in U.S. Pat. No. 5,143,854; PCT Patent Publication No.90/15070, published Dec. 13, 1990; U.S. patent application Ser. No.07/624,120, filed Dec. 6, 1990; Fodor, et al., Science, 251:767-773(February/1991); Dower, et al., Ann. Rep. Med. Chem., 26:271-180 (1991);and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991.

Using the procedures described above, random peptides were generallydesigned to have a defined number of amino acid residues in length(e.g., 12). To generate the collection of oligonucleotides encoding therandom peptides, the codon motif (NNK)x, where N is nucleotide A, C, G,or T (equimolar; depending on the methodology employed, othernucleotides can be employed), K is G or T (equimolar), and x is aninteger corresponding to the number of amino acids in the peptide (e.g.,12) was used to specify any one of the 32 possible codons resulting fromthe NNK motif: 1 for each of 12 amino acids, 2 for each of 5 aminoacids, 3 for each of 3 amino acids, and only one of the three stopcodons. Thus, the NNK motif encodes all of the amino acids, encodes onlyone stop codon, and reduces codon bias.

In the systems employed, the random peptides were presented either onthe surface of a phage particle, as part of a fusion protein comprisingeither the pIII or the pVIII coat protein of a phage fd derivative(peptides on phage) or as a fusion protein with the LacI peptide fusionprotein bound to a plasmid (peptides on plasmids).

The phage or plasmids, including the DNA encoding the peptides, wereidentified and isolated by an affinity enrichment process usingimmobilized TPO-R. The affinity enrichment process, sometimes called“panning,” involves multiple rounds of incubating the phage, plasmids,or polysomes with the immobilized receptor, collecting the phage,plasmids, or polysomes that bind to the receptor (along with theaccompanying DNA or mRNA), and producing more of the phage or plasmids(along with the accompanying LacI-peptide fusion protein) collected. Theextracellular domain (ECD) of the TPO-R typically was used duringpanning.

After several rounds of affinity enrichment, the phage or plasmids andaccompanying peptides were examined by ELISA to determine if thepeptides bind specifically to TPO-R. This assay was carried outsimilarly to the procedures used in the affinity enrichment process,except that after removing unbound phage, the wells were typicallytreated with rabbit anti-phage antibody, then with alkaline phosphatase(AP)-conjugated goat anti-rabbit antibody. The amount of alkalinephosphatase in each well was determined by standard methods. A similarELISA procedure for use in the peptides on plasmids system is describedin detail below.

By comparing test wells with control wells (no receptor), one candetermine whether the fusion proteins bind to the receptor specifically.The phage pools found to bind to TPO-R were screened in a colony liftprobing format using radiolabelled monovalent receptor. This probe canbe produced using protein kinase A to phosphorylate a peptide sequencefused to the C-terminus of the soluble receptor. The “engineered” formof the TPO receptor is then expressed in host cells, typically CHOcells. Following PI-PLC harvest of the receptors, the receptor wastested for binding to TPO or TPO-R specific phage clones. The receptoris then labeled to high specific activity with 33 P for use as amonovalent probe to identify high affinity ligands using colony lifts.

Peptides found to bind specifically to the receptor were thensynthesized as the free peptide (e.g., no phage) and tested in ablocking assay. The blocking assay was carried out in similar fashion tothe ELISA, except that TPO or a reference peptide was added to the wellsbefore the fusion protein (the control wells were of two types: (1) noreceptor; and (2) no TPO or reference peptide). Fusion proteins forwhich the binding to the receptor was blocked by TPO or the referencepeptide contain peptides in the random peptide portion that arepreferred compounds of the invention.

TPO-R, as well as its extracellular domain, were produced in recombinanthost cells. One useful form of TPO-R is constructed by expressing theprotein as a soluble protein in baculovirus transformed host cells usingstandard methods; another useful form is constructed with a signalpeptide for protein secretion and for glycophospholipid membrane anchorattachment. This form of anchor attachment is called “PIG-tailing”. SeeCaras, et al., Science, 243:1196-1198 (1989) and Lin, et al., Science,249:677-679 (1990).

Using the PIG-tailing system, one can cleave the receptor from thesurface of the cells expressing the receptor (e.g., transformed CHOcells selected for high level expression of receptor with a cell sorter)with phospholipase C. The cleaved receptor still comprises a carboxyterminal sequence of amino acids, called the “HPAP tail”, from thesignal protein for membrane attachment and can be immobilized withoutfurther purification. The recombinant receptor protein can beimmobilized by coating the wells of microtiter plates with an anti-HPAPtail antibody (Ab 179 or MAb 179), blocking non-specific binding withbovine serum albumin (BSA) in PBS, and then binding cleaved recombinantreceptor to the antibody. Using this procedure, one should perform theimmobilization reaction in varying concentrations of receptor todetermine the optimum amount for a given preparation, because differentpreparations of recombinant protein often contain different amounts ofthe desired protein. In addition, one should ensure that theimmobilizing antibody is completely blocked (with TPO or some otherblocking compound) during the affinity enrichment process. Otherwise,unblocked antibody can bind undesired phage during the affinityenrichment procedure. One can use peptides that bind to the immobilizingantibody to block unbound sites that remain after receptorimmobilization to avoid this problem or one can simply immobilize thereceptor directly to the wells of microtiter plates, without the aid ofan immobilizing antibody. See U.S. patent application Ser. No.07/947,339, filed Sep. 18, 1992, incorporated herein by reference.

When using random peptide generation systems that allow for multivalentligand-receptor interaction, one must recognize that the density of theimmobilized receptor is an important factor in determining the affinityof the ligands that can bind to the immobilized receptor. At higherreceptor densities (e.g., each anti-receptor antibody-coated welltreated with 0.25 to 0.5 mg of receptor), multivalent binding is morelikely to occur than at lower receptor densities (e.g., eachanti-receptor antibody-coated well treated with 0.5 to 1 ng of thereceptor). If multivalent binding is occurring, then one will be morelikely to isolate ligands with relatively lower affinity, unless oneuses high densities of immobilized receptor to identify lead compoundsand uses lower receptor densities to isolate higher affinity derivativecompounds.

To discriminate among higher affinity peptides, a monovalent receptorprobe frequently is used. This probe can be produced using proteinkinase A to phosphorylate a peptide sequence fused to the C-terminus ofthe soluble receptor. The “engineered” form of the TPO receptor is thenexpressed in host cells, typically CHO cells. Following PI-PLC harvestof the receptors, the receptor was tested for binding to TPO or TPO-Rspecific phage clones. The receptor is then labeled to high specificactivity with 33 P for use as a monovalent probe to identify highaffinity ligands using colony lifts.

Preferred screening methods to facilitate identification of peptideswhich bind TPO-R involve first identifying lead peptides which bind tothe extracellular domain of the receptor and then making other peptideswhich resemble the lead peptides. Specifically, using a pIII orpVIII-based peptides on phage system, a random library can be screenedto discover a phage that presents a peptide that binds to TPO-R. Thephage DNAs are sequenced to determine the sequences of the peptidesdisplayed on the surface of the phages.

Clones capable of specific binding to the TPO-R were identified from arandom linear 10-mer pVIII library and a random cyclic 10-mer and 12-merpVIII libraries. The sequences of these peptides serve as the basis forthe construction of other peptide libraries designed to contain a highfrequency of derivatives of the initially identified peptides. Theselibraries can be synthesized so as to favor the production of peptidesthat differ from the binding peptide in only a few residues. Thisapproach involves the synthesis of an oligonucleotide with the bindingpeptide coding sequence, except that rather than using pure preparationsof each of the four nucleoside triphosphates in the synthesis, one usesmixtures of the four nucleoside triphosphates (i.e., 55% of the“correct” nucleotide, and 15% each of the other three nucleotides is onepreferred mixture for this purpose and 70% of the “correct” nucleotideand 10% of each of the other three nucleotides is another preferredmixture for this purpose) so as to generate derivatives of the bindingpeptide coding sequence.

A variety of strategies were used to derivatize the lead peptides bymaking “mutagenesis on a theme” libraries. These included a pVIIIphagemid mutagenesis library based on the consensus sequence mutagenizedat 70:10:10:10 frequency and extended on each terminus with randomresidues to produce clones.

The “peptides on plasmids” techniques was also used for peptidescreening and mutagenesis studies and is described in greater detail inU.S. Pat. No. 5,338,665, which is incorporated herein by reference forall purposes. According to this approach, random peptides are fused atthe C-terminus of LacI through expression from a plasmid vector carryingthe fusion gene. Linkage of the LacI-peptide fusion to its encoding DNAoccurs via the lacO sequences on the plasmid, forming a stablepeptide-LacI-plasmid complex that can be screened by affinitypurification (panning) on an immobilized receptor. The plasmids thusisolated can then be reintroduced into E. coli by electroporation toamplify the selected population for additional rounds of screening, orfor the examination of individual clones.

In addition, random peptide screening and mutagenesis studies can beperformed using a modified C-terminal Lac-I display system in whichdisplay valency was reduced (“headpiece dimer” display system). Thelibraries were screened and the resulting DNA inserts can be cloned as apool into a maltose binding protein (MBP) vector allowing theirexpression as a C-terminal fusion protein. Crude cell lysates fromrandomly picked individual MBP fusion clones can then be assayed forTPO-R binding in an ELISA format, as discussed above.

Peptide mutagenesis studies can also be conducted using the polysomedisplay system, as described in U.S. patent application Ser. No.08/300,262, filed Sep. 2, 1994, which is a continuation-in-partapplication based on U.S. patent application Ser. No. 08/144,775, filedOct. 29, 1993 and PCT WO 95/11992, each of which is incorporated hereinby references for all purposes.

It was found that the core peptide can comprises a sequence of aminoacids: (SEQ ID NO:2) X₉ X₈ G X₁ X₂ X₃X₄ X₅ X₆ X₇, where X₆ may beβ-(1-napthy)alanine and where X₉ is A, C, E, G, I, L, M, P, R, Q, S, T,or V; and X₈ is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, X₉is A or I; and X₈ is D, E, or K. Further X₁ is C, L, M, P, Q, V; X₂ isF, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S, V or W; X₄ isany of the 20 genetically coded L-amino acids; X₅ is A, D, E, G, K, M,Q, R, S, T, V or Y; and X₇ is C, G, I, K, L, M, N, R or V.

However, as described further below, it has been surprisingly found thatby replacing X₆ with β-(2-napthy)alanine, the compound provides enhancedproperties over the compound containing β-(1-napthy)alanine.Accordingly, a particularly preferred peptide includes the amino acidsequence (SEQ ID NO:3): I E G P T L R Q (2-Nal) L A A RA.

In another embodiment, the peptide compounds of the present inventionare preferably dimerized or oligomerized to increase the affinity and/oractivity of the compounds. An example of a preferred dimerized peptidecompound includes, but is not limited to, the following:

Where X₁₀ is a sarcosine or β-alanine residue (SEQ ID NO:4). It shouldbe noted that one X₁₀ residue can be sarcosine and the other residue canbe β-alanine. The above structure can also be represented by thefollowing: (H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH2.

Peptides and peptidomimetics having an IC₅₀ of greater than about 100 mMlack sufficient binding to permit use in either the diagnostic ortherapeutic aspects of this invention. Preferably, for diagnosticpurposes, the peptides and peptidomimetics have an IC₅₀ of about 2 mM orless and, for pharmaceutical purposes, the peptides and peptidomimeticshave an IC₅₀ of about 100 μM or less.

FIG. 1 compares the activity of three different batches of un-PEGylatedIEGPTLRQ(2-Nal)LAAR with un-PEGylated IEGPTLRQ(1-Nal)LAAR using standardrelative luminescent units assay techniques. The assay employs murinecells engineered to stably express the human TPO receptor and aluciferase reporter construct driven by the fos promoter. As shown fromFIG. 1, the activity is similar for each compound.

FIG. 2 compares the activity of several different PEGylated peptides(pegylation of the compounds of the present invention is described inmore detail below). Both of the PEGylated IEGPTLRQ(1-Nal)LAAR compoundsshow high activity with essentially the same level of activity as theun-PEGylated peptide. The remaining lines illustrate the activity ofdifferent PEGylated batches of dimerized IEGPTLRQ(2-Nal)LAAR. As shownby FIG. 2, in this model, the latter have less activity relative to thePEGylated IEGPTLRQ(1-Nal)LAAR compounds.

FIG. 3 demonstrates the relative potency of a PEGylated peptidecontaining β-(1-napthyl)alanine) and the PEGylated peptide containingβ-(2-naphthyl)alanine. Through a rat model, FIG. 3 shows the in-vivochange in platelet counts after administration of dimerized PEGylatedα-(2-naphthyl)alanine and β-(1-napthyl)alanine. As shown by FIG. 3, thehighest dose of the PEGylated β-(2-naphthyl)alanine material has thesame activity as the lowest dose of the PEGylated β-(1-napthyl)alanine.A less potent compound may provide a less drastic stimulus to the targetcell, which could reduce the risk of side effects caused byoverstimulation of the target cell, such as exacerbated thrombocytopeniafollowing subsequent cycle of chemotherapy.

FIGS. 4 and 5 show the results of a head-to-head dose response study ofa PEGylated peptide containing β-(1-napthyl)alanine) and the PEGylatedpeptide containing β-(2-naphthyl)alanine in normal mice. FIG. 4 showsincreases in platelet levels and FIG. 5 shows Mean Platelet Volume six(6) days following treatment. The dose range was from 10 to 3000 ug/kg.Both compounds increased the number of circulating platelets in adose-dependent manner with increases relative to the control groupobserved at doses as low as 30 ug/kg for both compounds. At the maximalresponse, these compounds elevated platelet counts to levels that wereup to 4-fold greater than control values. The dose-response curves forthese compounds were very similar indicating that in this model therewas essentially no difference between the two test articles based onthese endpoints.

IV. Preparation of Peptides and Peptide Mimetics

A. Solid Phase Synthesis

The peptides of the invention can be prepared by classical methods knownin the art, for example, by using standard solid phase techniques. Thestandard methods include exclusive solid phase synthesis, partial solidphase synthesis methods, fragment condensation, classical solutionsynthesis, and even by recombinant DNA technology. See, e.g.,Merrifield, J. Am. Chem. Soc., 85:2149 (1963), incorporated herein byreference. On solid phase, the synthesis is typically commenced from theC-terminal end of the peptide using an alpha-amino protected resin. Asuitable starting material can be prepared, for instance, by attachingthe required alpha-amino acid to a chloromethylated resin, ahydroxymethyl resin, or a benzhydrylamine resin. One suchchloromethylated resin is sold under the tradename BIO-BEADS SX-1 by BioRad Laboratories, Richmond, Calif., and the preparation of thehydroxymethyl resin is described by Bodonszky, et al., Chem. Ind.(London), 38:1597 (1966). The benzhydrylamine (BHA) resin has beendescribed by Pietta and Marshall, Chem. Commn., 650 (1970) and iscommercially available from Beckman Instruments, Inc., Palo Alto,Calif., in the hydrochloride form.

Thus, the compounds of the invention can be prepared by coupling analpha-amino protected amino acid to the chloromethylated resin with theaid of, for example, cesium bicarbonate catalyst, according to themethod described by Gisin, Helv. Chim. Acta., 56:1467 (1973). After theinitial coupling, the alpha-amino protecting group is removed by achoice of reagents including trifluoroacetic acid (TFA) or hydrochloricacid (HCl) solutions in organic solvents at room temperature.

The alpha-amino protecting groups are those known to be useful in theart of stepwise synthesis of peptides. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane typeprotecting groups (e.g. benzyloxycarboyl (Cbz) and substituted Cbz),aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protectinggroups (e.g., benzyl, triphenylmethyl). Boc and Fmoc are preferredprotecting groups. The side chain protecting group remains intact duringcoupling and is not split off during the deprotection of theamino-terminus protecting group or during coupling. The side chainprotecting group must be removable upon the completion of the synthesisof the final peptide and under reaction conditions that will not alterthe target peptide.

The side chain protecting groups for Tyr include tetrahydropyranyl,tert-butyl, trityl, benzyl, Cbz, Z—Br—Cbz, and 2,5-dichlorobenzyl. Theside chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl,methyl, ethyl, and cyclohexyl. The side chain protecting groups for Thrand Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl,2,6-dichlorobenzyl, and Cbz. The side chain protecting group for Thr andSer is benzyl. The side chain protecting groups for Arg include nitro,Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc.The side chain protecting groups for Lys include Cbz,2-chlorobenzyloxycarbonyl (2-Cl—Cbz), 2-bromobenzyloxycarbonyl(2-BrCbz), Tos, or Boc.

After removal of the alpha-amino protecting group, the remainingprotected amino acids are coupled stepwise in the desired order. Anexcess of each protected amino acid is generally used with anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride (CH₂Cl₂), dimethylformamide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagentsuch as trifluoroacetic acid or hydrogen fluoride (HF), which not onlycleaves the peptide from the resin, but also cleaves all remaining sidechain protecting groups. When the chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides.

These solid phase peptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Using the “encoded synthetic library” or “very large scale immobilizedpolymer synthesis” system described in U.S. patent application Ser. No.07/492,462, filed Mar. 7, 1990; Ser. No. 07/624,120, filed Dec. 6, 1990;and Ser. No. 07/805,727, filed Dec. 6, 1991; one can not only determinethe minimum size of a peptide with such activity, one can also make allof the peptides that form the group of peptides that differ from thepreferred motif (or the minimum size of that motif) in one, two, or moreresidues. This collection of peptides can then be screened for abilityto bind to TPO-R. This immobilized polymer synthesis system or otherpeptide synthesis methods can also be used to synthesize truncationanalogs and deletion analogs and combinations of truncation and deletionanalogs of all of the peptide compounds of the invention.

B. Synthetic Amino Acids

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of any of thecompounds of the invention. For instance, naphthylalanine can besubstituted for tryptophan, facilitating synthesis. Other syntheticamino acids that can be substituted into the peptides of the presentinvention include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, d aminoacids such as L-d-hydroxylysyl and D-d-methylalanyl, L-α-methylalanyl, βamino acids, and isoquinolyl. D amino acids and non-naturally occurringsynthetic amino acids can also be incorporated into the peptides of thepresent invention (see, e.g., Roberts, et al., Unusual Amino/Acids inPeptide Synthesis, 5(6):341-449 (1983)).

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains, for instance with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered hetereocyclic. Inparticular, proline analogs in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino),oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g. 1-piperidyl,piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g. 1-pyrrolidinyl),pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl(e.g. thiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

One can also readily modify the peptides of the instant invention byphosphorylation (see, e.g., W. Bannwarth, et al., Biorganic andMedicinal Chemistry Letters, 6(17):2141-2146 (1996)), and other methodsfor making peptide derivatives of the compounds of the present inventionare described in Hruby, et al., Biochem. J., 268(2):249-262 (1990).Thus, the peptide compounds of the invention also serve as a basis toprepare peptide mimetics with similar biological activity.

C. Terminal Modifications

Those of skill in the art recognize that a variety of techniques areavailable for constructing peptide mimetics with the same or similardesired biological activity as the corresponding peptide compound butwith more favorable activity than the peptide with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis.See, for example, Morgan, et al., Ann. Rep. Med. Chem., 24:243-252(1989). The following describes methods for preparing peptide mimeticsmodified at the N-terminal amino group, the C-terminal carboxyl group,and/or changing one or more of the amido linkages in the peptide to anon-amido linkage. It being understood that two or more suchmodifications can be coupled in one peptide mimetic structure (e.g.,modification at the C-terminal carboxyl group and inclusion of a—CH₂-carbamate linkage between two amino acids in the peptide).

1. N-terminal Modifications

The peptides typically are synthesized as the free acid but, as notedabove, could be readily prepared as the amide or ester. One can alsomodify the amino and/or carboxy terminus of the peptide compounds of theinvention to produce other compounds of the invention. Amino terminusmodifications include methylation, acetylation, adding abenzyloxycarbonyl group, or blocking the amino terminus with anyblocking group containing a carboxylate functionality defined by RCOO—,where R is selected from the group consisting of naphthyl, acridinyl,steroidyl, and similar groups. Carboxy terminus modifications includereplacing the free acid with a carboxamide group or forming a cycliclactam at the carboxy terminus to introduce structural constraints.

Amino terminus modifications are as recited above and includealkylating, acetylating, adding a carbobenzoyl group, forming asuccinimide group, etc. (See, e.g., Murray, et al., Burger's MedicinalChemistry and Drug Discovery, 5th ed., Vol. 1, Manfred E. Wolf, ed.,John Wiley and Sons, Inc. (1995).) Specifically, the N-terminal aminogroup can then be reacted as follows:

-   (a) to form an amide group of the formula RC(O)NH— where R is as    defined above by reaction with an acid halide or symmetric    anhydride. Typically, the reaction can be conducted by contacting    about equimolar or excess amounts (e.g., about 5 equivalents) of an    acid halide to the peptide in an inert diluent (e.g.,    dichloromethane) preferably containing an excess (e.g., about 10    equivalents) of a tertiary amine, such as diisopropylethylamine, to    scavenge the acid generated during reaction. Reaction conditions are    otherwise conventional (e.g., room temperature for 30 minutes).    Alkylation of the terminal amino to provide for a lower alkyl    N-substitution followed by reaction with an acid halide as described    above will provide for N-alkyl amide group of the formula RC(O)NR—;-   (b) to form a succinimide group by reaction with succinic anhydride.    As before, an approximately equimolar amount or an excess of    succinic anhydride (e.g., about 5 equivalents) can be employed and    the amino group is converted to the succinimide by methods well    known in the art including the use of an excess (e.g., ten    equivalents) of a tertiary amine such as diisopropylethylamine in a    suitable inert solvent (e.g., dichloromethane). See, for example,    Wollenberg, et al., U.S. Pat. No. 4,612,132 which is incorporated    herein by reference in its entirety. It is understood that the    succinic group can be substituted with, for example, alkyl or —SR    substituents which are prepared in a conventional manner to provide    for substituted succinimide at the N-terminus of the peptide. Such    alkyl substituents are prepared by reaction of a lower olefin with    maleic anhydride in the manner described by Wollenberg, et al.,    supra and —SR substituents are prepared by reaction of RSH with    maleic anhydride where R is as defined above;-   (c) to form a benzyloxycarbonyl-NH—or a substituted    benzyloxycarbonyl-NH—group by reaction with approximately an    equivalent amount or an excess of CBZ—Cl (i.e., benzyloxycarbonyl    chloride) or a substituted CBZ—Cl in a suitable inert diluent (e.g.,    dichloromethane) preferably containing a tertiary amine to scavenge    the acid generated during the reaction;-   (d) to form a sulfonamide group by reaction with an equivalent    amount or an excess (e.g., 5 equivalents) of R—S(O)₂ Cl in a    suitable inert diluent (dichloromethane) to convert the terminal    amine into a sulfonamide where R is as defined above. Preferably,    the inert diluent contains excess tertiary amine (e.g., ten    equivalents) such as diisopropylethylamine, to scavenge the acid    generated during reaction. Reaction conditions are otherwise    conventional (e.g., room temperature for 30 minutes);-   (e) to form a carbamate group by reaction with an equivalent amount    or an excess (e.g., 5 equivalents) of R—OC(O)Cl or    R—OC(O)OC₆H₄-p-NO₂ in a suitable inert diluent (e.g.,    dichloromethane) to convert the terminal amine into a carbamate    where R is as defined above. Preferably, the inert diluent contains    an excess (e.g., about 10 equivalents) of a tertiary amine, such as    diisopropylethylamine, to scavenge any acid generated during    reaction. Reaction conditions are otherwise conventional (e.g., room    temperature for 30 minutes); and-   (f) to form a urea group by reaction with an equivalent amount or an    excess (e.g., 5 equivalents) of R—N═C═O in a suitable inert diluent    (e.g., dichloromethane) to convert the terminal amine into a urea    (i.e., RNHC(O)NH—) group where R is as defined above. Preferably,    the inert diluent contains an excess (e.g., about 10 equivalents) of    a tertiary amine, such as diisopropylethylamine. Reaction conditions    are otherwise conventional (e.g., room temperature for about 30    minutes).

2. C-Terminal Modifications

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by an ester (i.e., —C(O)OR where R is as defined above), theresins used to prepare the peptide acids are employed, and the sidechain protected peptide is cleaved with base and the appropriatealcohol, e.g., methanol. Side chain protecting groups are then removedin the usual fashion by treatment with hydrogen fluoride to obtain thedesired ester.

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by the amide —C(O)NR³, R⁴, a benzhydrylamine resin is used asthe solid support for peptide synthesis. Upon completion of thesynthesis, hydrogen fluoride treatment to release the peptide from thesupport results directly in the free peptide amide (i.e., the C-terminusis —C(O)NH₂). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR¹where R and R¹ are as defined above). Side chain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride to givethe free amides, alkylamides, or dialkylamides.

One can also cyclize the peptides of the invention, or incorporate adesamino or descarboxy residue at the terminii of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds of the present invention includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

In addition to the foregoing N-terminal and C-terminal modifications,the peptide compounds of the invention, including peptidomimetics, canadvantageously be modified with or covalently coupled to one or more ofa variety of hydrophilic polymers. It has been found that when thepeptide compounds are derivatized with a hydrophilic polymer, theirsolubility and circulation half-lives are increased and theirimmunogenicity is masked. Quite surprisingly, the foregoing can beaccomplished with little, if any, diminishment in their bindingactivity. Nonproteinaceous polymers suitable for use in accordance withthe present invention include, but are not limited to, polyalkylethersas exemplified by polyethylene glycol and polypropylene glycol,polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc. Generally, such hydrophilic polymers have anaverage molecular weight ranging from about 500 to about 100,000daltons, more preferably from about 2,000 to about 40,000 daltons and,even more preferably, from about 5,000 to about 20,000 daltons. Inpreferred embodiments, such hydrophilic polymers have an averagemolecular weights of about 5,000 daltons, 10,000 daltons and 20,000daltons.

The peptide compounds of the invention can be derivatized with orcoupled to such polymers using any of the methods set forth inZallipsky, S., Bioconjugate Chem., 6:150-165 (1995); Monfardini, C, etal., Bioconjugate Chem., 6:62-69 (1995); U.S. Pat. No. 4,640,835; U.S.Pat. No. 4,496,689; U.S. Pat. No. 4,301,144; U.S. Pat. No. 4,670,417;U.S. Pat. No. 4,791,192; U.S. Pat. No. 4,179,337 or WO 95/34326, all ofwhich are incorporated by reference in their entirety herein.

In a presently preferred embodiment, the peptide compounds of thepresent invention are derivatized with polyethylene glycol (PEG). PEG isa linear, water-soluble polymer of ethylene oxide repeating units withtwo terminal hydroxyl groups. PEGs are classified by their molecularweights which typically range from about 500 daltons to about 40,000daltons. In a presently preferred embodiment, the PEGs employed havemolecular weights ranging from 5,000 daltons to about 20,000 daltons.PEGs coupled to the peptide compounds of the present invention can beeither branched or unbranched. (See, e.g., Monfardini, C., et al.,Bioconjugate Chem., 6:62-69 (1995)). PEGs are commercially availablefrom Shearwater Polymers, Inc. (Huntsville, Ala.), Sigma Chemical Co.and other companies. Such PEGs include, but are not limited to,monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, in one exemplar embodiment, the hydrophilic polymer which isemployed, e.g., PEG, is preferably capped at one end by an unreactivegroup such as a methoxy or ethoxy group. Thereafter, the polymer isactivated at the other end by reaction with a suitable activating agent,such as cyanuric halides (e.g., cyanuric chloride, bromide or fluoride),diimadozle, an anhydride reagent (e.g., a dihalosuccinic anhydride, suchas dibromosuccinic anhydride), acyl azide, p-diazoiumbenzyl ether,3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activatedpolymer is then reacted with a peptide compound of the present inventionto produce a peptide compound derivatized with a polymer. Alternatively,a functional group in the peptide compounds of the invention can beactivated for reaction with the polymer, or the two groups can be joinedin a concerted coupling reaction using known coupling methods. It willbe readily appreciated that the peptide compounds of the invention canbe derivatized with PEG using a myriad of other reaction schemes knownto and used by those of skill in the art.

In addition to derivatizing the peptide compounds of this invention witha hydrophilic polymer (e.g., PEG), other small peptides, e.g., otherpeptides or ligands that bind to a receptor, can also be derivatizedwith such hydrophilic polymers with little, if any, loss in biologicalactivity (e.g., binding activity, agonist activity, antagonist activity,etc.). It has been found that when these small peptides are derivatizedwith a hydrophlilic polymer, their solubility and circulation half-livesare increased and their immunogenicity is decreased. Again, quitesurprisingly, the foregoing can be accomplished with little, if any,loss in biological activity. In fat, in preferred embodiments, thederivatized peptides have an activity that is 0.1 to 0.01-fold that ofthe unmodified peptides. In more preferred embodiments, the derivatizedpeptides have an activity that is 0.1 to 1-fold that of the unmodifiedpeptides. In even more preferred embodiments, the derivatized peptideshave an activity that is greater than the unmodified peptides.

Peptides suitable for use in this embodiment generally include thosepeptides, i.e., ligands, that bind to a receptor, such as the TPO, EPO,IL-1, G-CSF and IL-5 receptors; the hematopoietic growth factorreceptors; the cytokine receptors; the G-protein-linked receptors; thecell surface receptors, etc. Such peptides typically comprise about 150amino acid residues or less and, more preferably, about 100 amino acidresidues or less (e.g., .about.10-12 kDa). Hydrophilic polymers suitablefor use in the present invention include, but are not limited to,polyalkylethers as exemplified by polyethylene glycol and polypropyleneglycol, polylactic acid, polyglycolic acid, polyoxyalkenes,polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulosederivatives, dextran and dextran derivatives, etc. Generally, suchhydrophilic polymers have an average molecular weight ranging from about500 to about 100,000 daltons, more preferably from about 2,000 to about40,000 daltons and, even more preferably, from about 5,000 to about20,000 daltons. In preferred embodiments, such hydrophilic polymers havean average molecular weights of about 5,000 daltons, 10,000 daltons and20,000 daltons. The peptide compounds of this invention can bederivatized with using the methods described above and in the citedreferences.

D. Backbone Modifications

Other methods for making peptide derivatives of the compounds of thepresent invention are described in Hruby, et al., Biochem J.,268(2):249-262 (1990), incorporated herein by reference. Thus, thepeptide compounds of the invention also serve as structural models fornon-peptidic compounds with similar biological activity. Those of skillin the art recognize that a variety of techniques are available forconstructing compounds with the same or similar desired biologicalactivity as the lead peptide compound but with more favorable activitythan the lead with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis. See Morgan, et al., Ann. Rep. Med. Chem.,24:243-252 (1989), incorporated herein by reference. These techniquesinclude replacing the peptide backbone with a backbone composed ofphosphonates, amidates, carbamates, sulfonamides, secondary amines, andN-methylamino acids.

Suitable reagents include, for example, amino acid analogues wherein thecarboxyl group of the amino acid has been replaced with a moietysuitable for forming one of the above linkages.

Similarly, replacement of an amido linkage in the peptide with aphosphonate linkage can be achieved in the manner set forth in U.S.patent application Ser. Nos. 07/943,805, 08/081,577, and 08/119,700, thedisclosures of which are incorporated herein by reference in theirentirety.

Replacement of an amido linkage in the peptide with a urea linkage canbe achieved in the manner set forth in U.S. patent application Ser. No.08/147,805 which application is incorporated herein by reference in itsentirety.

E. Disulfide Bond Formation

The compounds of the present invention may exist in a cyclized form withan intramolecular disulfide bond between the thiol groups of thecysteines, if present. Alternatively, an intermolecular disulfide bondbetween the thiol groups of the cysteines can be produced to yield adimeric (or higher oligomeric) compound. One or more of the cysteineresidues may also be substituted with a homocysteine.

V. Utility

The compounds of the invention are useful in vitro as unique tools forunderstanding the biological role of TPO, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of TPO and the receptor binding process. The presentcompounds are also useful in the development of other compounds thatbind to and activate the TPO-R, because the present compounds provideimportant information on the relationship between structure and activitythat should facilitate such development.

The compounds are also useful as competitive binders in assays to screenfor new TPO receptor agonists. In such assay embodiments, the compoundsof the invention can be used without modification or can be modified ina variety of ways; for example, by labeling, such as covalently ornon-covalently joining a moiety which directly or indirectly provides adetectable signal. In any of these assays, the materials thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups such as: radiolabels such as 125 I, enzymes (U.S.Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, andfluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring thechange in fluorescence intensity, wavelength shift, or fluorescencepolarization. Possibilities for indirect labeling include biotinylationof one constituent followed by binding to avidin coupled to one of theabove label groups. The compounds may also include spacers or linkers incases where the compounds are to be attached to a solid support.

Moreover, based on their ability to bind to the TPO receptor, thepeptides of the present invention can be used as reagents for detectingTPO receptors on living cells, fixed cells, in biological fluids, intissue homogenates, in purified, natural biological materials, etc. Forexample, by labelling such peptides, one can identify cells having TPO-Ron their surfaces. In addition, based on their ability to bind the TPOreceptor, the peptides of the present invention can be used in in situstaining, FACS (fluorescence-activated cell sorting), Western blotting,ELISA, etc. In addition, based on their ability to bind to the TPOreceptor, the peptides of the present invention can be used in receptorpurification, or in purifying cells expressing TPO receptors on the cellsurface (or inside permeabilized cells).

The compounds of the present invention can also be utilized ascommercial reagents for various medical research and diagnostic uses.Such uses include but are not limited to: (1) use as a calibrationstandard for quantitating the activities of candidate TPO agonists in avariety of functional assays; (2) use to maintain the proliferation andgrowth of TPO-dependent cell lines; (3) use in structural analysis ofthe TPO-receptor through co-crystallization; (4) use to investigate themechanism of TPO signal transduction/receptor activation; and (5) otherresearch and diagnostic applications wherein the TPO-receptor ispreferably activated or such activation is conveniently calibratedagainst a known quantity of a TPO agonist, and the like.

The compounds of the present invention can be used for the in vitroexpansion of megakaryocytes and their committed progenitors, both inconjunction with additional cytokines or on their own. See, e.g.,DiGiusto, et al., PCT Publication No. 95/05843, which is incorporatedherein by reference. Chemotherapy and radiation therapies causethrombocytopenia by killing the rapidly dividing, more mature populationof megakaryocytes. However, these therapeutic treatments can also reducethe number and viability of the immature, less mitotically activemegakaryocyte precursor cells. Thus, amelioration of thethrombocytopenia by TPO or the compounds of the present invention can behastened by infusing patients post chemotherapy or radiation therapywith a population of his or her own cells enriched for megakaryocytesand immature precursors by in vitro culture.

The compounds of the invention can also be administered to warm bloodedanimals, including humans, to activate the TPO-R in vivo. Thus, thepresent invention encompasses methods for therapeutic treatment of TPOrelated disorders that comprise administering a compound of theinvention in amounts sufficient to mimic the effect of TPO on TPO-R invivo. For example, the peptides and compounds of the invention can beadministered to treat a variety of hematological disorders, includingbut not limited to platelet disorders and thrombocytopenia, particularlywhen associated with bone marrow transfusions, radiation therapy, andchemotherapy.

In some embodiments of the invention, TPO antagonists are preferablyfirst administered to patients undergoing chemotherapy or radiationtherapy, followed by administration of the TPO agonists of theinvention.

The activity of the compounds of the present invention can be evaluatedeither in vitro or in vivo in one of the numerous models described inMcDonald, Am. J. of Pediatric Hematology/Oncology, 14:8-21 (1992), whichis incorporated herein by reference.

According to one embodiment, the compositions of the present inventionare useful for treating thrombocytopenia associated with bone marrowtransfusions, radiation therapy, or chemotherapy. The compoundstypically will be administered prophylactically prior to chemotherapy,radiation therapy, or bone marrow transplant or after such exposure.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising, as an active ingredient, at least one of thepeptides or peptide mimetics of the invention in association with apharmaceutical carrier or diluent. The compounds of this invention canbe administered by oral, pulmonary, parental (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation), transdermal, nasal, vaginal, rectal, orsublingual routes of administration and can be formulated in dosageforms appropriate for each route of administration. See, e.g.,Bernstein, et al., PCT Patent Publication No. WO 93/25221; Pitt, et al.,PCT Patent Publication No. WO 94/17784; and Pitt, et al., EuropeanPatent Application 613,683, each of which is incorporated herein byreference.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parental administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

The compositions containing the compounds can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective dose”. Amounts effective for this use will depend on theseverity of the disease and the weight and general state of the patient.

The compositions of the invention can also be microencapsulated by, forexample, the method of Tice and Bibi (in Treatise on Controlled DrugDelivery, ed. A. Kydonieus, Marcel Dekker, New York (1992), pp.315-339).

In prophylactic applications, compositions containing the compounds ofthe invention are administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be a“prophylactically effective dose”. In this use, the precise amountsagain depend on the patient's state of health and weight.

The quantities of the TPO agonist necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds),Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8thed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 7thEd., Mack Publishing Co., Easton, Pa. (1985); each of which is herebyincorporated by reference.

The peptides and peptide mimetics of this invention are effective intreating TPO mediated conditions when administered at a dosage range offrom about 0.001 mg to about 10 mg/kg of body weight per day. Thespecific dose employed is regulated by the particular condition beingtreated, the route of administration as well as by the judgement of theattending clinician depending upon factors such as the severity of thecondition, the age and general condition of the patient, and the like.

Although only preferred embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of the invention are possible without departing from thespirit and intended scope of the invention.

EXAMPLE 1 Solid Phase Peptide Synthesis

Various peptides of the invention were synthesized using the Merrifieldsolid phase synthesis techniques (see Steward and Young, Solid PhasePeptide Synthesis, 2d. edition, Pierce Chemical, Rockford, Ill. (1984)and Merrifield, J. Am. Chem. Soc., 85:2149 (1963)) or an AppliedBiosystems Inc. Model 431A or 433A peptide synthesizer. The peptideswere assembled using standard protocols of the Applied Biosystems Inc.Synth Assist.TM. 1.0.0 or Synth Assist.TM. 2.0.2. Each coupling wasperformed for 2.times.30 min. with HBTU(2-(1H-benzatriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) and HOBt (1-hydroxybenzotriazole).

The resin used was HMP resin (p-hydroxymethyl phenoxymethyl)polystyreneresin or PAL (Milligen/Biosearch), which is a cross-linked polystyreneresin with 5-(4′-Fmoc-aminomethyl-3,5′-dimethyoxyphenoxy)valeric acid asa linker. Use of PAL resin results in a carboxyl terminal amidefunctionality upon cleavage of the peptide from the resin. Uponcleavage, the HMP resin produces a carboxylic acid moiety at theC-terminus of the final product. Most reagents, resins, and protectedamino acids (free or on the resin) were purchased from Millipore orApplied Biosystems Inc.

The Fmoc group was used for amino protection during the couplingprocedure. Primary amine protection on amino acids was achieved withFmoc and side chain protection groups were t-butyl for serine, tyrosine,glutamic acid, and threonine; trityl for glutamine; Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl) for arginine;N-t-butyloxycarbonyl for tryptophan; N-trityl for histidine and S-tritylfor cysteine.

Removal of the peptides from the resin and simultaneous deprotection ofthe side chain functions were achieved by treatment with reagent K orslight modifications of it. Alternatively, in the synthesis of thosepeptides, with an amidated carboxyl terminus, the fully assembledpeptide was cleaved with a mixture of 90% trifluoroacetic acid, 5%ethanedithiol, and 5% water, initially at 4.degree. C., and graduallyincreasing to room temperature. The deprotected peptides wereprecipitated with diethyl ether. In all cases, purification was bypreparative, reverse-phase, high performance liquid chromatography on aC₁₈ bonded silica gel column with a gradient of acetonitrile/water in0.1% trifluoroacetic acid. The homogeneous peptides were characterizedby Fast Atom Bombardment mass spectrometry or electrospray massspectrometry and amino acid analysis when applicable.

In a preferred embodiment, the peptides of this invention are dimerizedusing standard synthetic procedures known to and used by those of skillin the art. Following these synthetic schemes, those of skill in the artcan readily prepare dimer peptide compounds in accordance with thepresent invention. In addition, it will be readily apparent to those ofskill in the art that the dimeric subunits can readily be linked usingknown methodologies and linkers.

EXAMPLE 2 Pegylation of the Peptides

A polypeptide of the present invention was dissolved in 100 mM bicine pH8.0 at a concentration of 10 mg/ml, added to a 1.25 fold molar excess ofpowdered PEG2 (commercially available from Shearwater Polymers, Inc.(Huntsville, Ala.)) and stirred at room temperature until the reactionwas complete, typically 1-2 hours. The reaction was monitored by reversephase HPLC using a 40-65% acetonitrile gradient with a YMC ODS AQcolumn. When the reaction was complete, the solution was added to asecond 1.25 molar excess of powdered PEG2 and the process was repeated 4times using a total of 5 moles of PEG2 for each mole of polypeptide. Thesolution was diluted 2 fold with PBS to reduce the viscosity and loadedonto a superdex 200 column (Pharmacia), previously equilibrated andeluted with PBS. Fractions from the size exclusion column were analyzedby reverse phase HPLC. Fractions containing di-PEG-polypeptide whicheluted prior to any mono-PEG-polypeptide were pooled and stored at 5degrees C. or lyophilized.

EXAMPLE 3 Pre-Clinical Animal Studies on the Thrombopoietic Activity ofTPO Compound No. 1

TPO Compound No. 1 is a TPO mimetic peptide discovered by screening TPOreceptor against phage display combinatorial peptide library followed bypeptide optimization. TPO Compound No. 1 does not share any sequencehomology with endogenous TPO, mitigating the risk of the formation ofantibodies cross-reacting with endogenous TPO. TPO Compound No. 1 wasPEGylated to reduce clearance and to further reduce antigenicity. ThisExample describes pre-clinical studies on the thrombopoietic activity ofTPO Compound No. 1 in an animal.

Normal male Wistar rats (source) were used for the studies. Otheranimals, such as dogs, mice, monkeys, etc. can also be used for thepre-clinical studies. All procedures involving animals were conducted inan animal facility fully accredited by the American Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC) and inaccordance with The Guide for the Care and Use of Laboratory Animals(NIH).

Normal male Wistar rats (10 weeks of age, 230 to 367 grams body weightrange at dosing) were treated with single intravenous doses of TPOCompound No. 1 at 30, 100 or 300 ug/kg (40 rats/group). At predose, 96,144, 192, 240, 288 and 312 hours post dose, approximately 0.5 mL ofblood was collected by puncture of the jugular vein of unanesthetizedrats (5 rats per time point, EDTA as anticoagulant) and platelet countswere assessed using a automated hematology analysis system. Animals werefasted overnight, with water available, prior to each sample collection.

Single intravenous doses of TPO Compound No. 1 (30, 100 or 300 μg/kg)resulted in an increased peripheral platelet count in normal male Wistarrats by the earliest post-dose assessment on Day 4 (FIG. 6). Plateletcounts were assessed every 2 days during the 2-week follow-up andcompared with the predose count. The 300 μg/kg dose induced the greatestincrease in platelet count, which returned to baseline by Day 14.

EXAMPLE 4 Phase I Clinical Studies on the Thrombopoietic Activity of TPOCompound No. 1

Phase I studies were conducted to investigate the tolerability,pharmacodynamics and pharmacokinetics of TPO Compound No. 1. Thisexample describes Phase I studies on TPO Compound No. 1 after a singleintravenous injection in healthy male volunteers. Phase I studies on TPOCompound No. 1 and other compounds according to the invention aftermultiple intravenous injection or other means of administration and/orto a patient in need of a treatment can be performed using protocolsknown to a person skilled in the art.

Forty volunteers were randomized to receive TPO Compound No. 1 orplacebo as a single i.v. bolus injection in a ratio of 6:2. Eightsubjects were randomized in 6:2 ratio to receive a single injection ofTPO Compound No. 1 or placebo, with a dose range of 0.375, 0.75, 1.5,2.25 or 3 μg/kg. The pharmacodynamic response of TPO Compound No. 1 wasmeasured as elevation in platelet counts. TPO Compound No. 1 levels weredetermined in platelet poor plasma using a validated enzyme-linkedimmunosorbent assay. Levels of endogenous TPO, EPO, IL-6 and IL-11 weremeasured at the indicated time points using standard immunoassays. Abiosensor immunoassay (BiaCore technology) was used for measuringantibody formation against the peptide moiety of TPO Compound No. 1. Theeffect on platelet function was measured by monitoring collagen-inducedplatelet aggregation at 4 hours and 12 days after TPO Compound No. 1administration.

PK analysis indicated dose-related kinetics of TPO Compound No. 1,although at doses of 0.75 μg/kg or lower, plasma concentrations of TPOCompound No. 1 were generally below the limit of quantification of 6.25ng/mL (FIG. 7). Four subjects in the 0.375 μg/kg dose group and onesubject in the 3.0 μg/kg TPO Compound No. 1 dose group had noquantifiable plasma levels. Mean C_(max) values ranged from 10.9 ng/mLat 0.75 μg/kg TPO Compound No. 1 to 61.7 ng/mL at 3.0 μg/kg TPO CompoundNo. 1 (Table 1). No PK data could be measured at 0.375 μg/kg i.v. of TPOCompound No. 1. The mean terminal half-life of TPO Compound No. 1 rangedfrom approximately 18 to 36 hours. The median t_(max) ranged from 0.09to 2 hours. The increase in C_(max) with increasing dose wasapproximately dose proportional, but there was an apparent increase inthe normalized AUC₀₋₂₄ value with increasing dose, suggesting a higherthan dose proportional increase. TABLE 1 Summary of PK analysis Cmaxt_(1/2) AUC_(oo) AUC₀₋₂₄ (ng/mL) (h) (ng · h/mL) (ng · h/mL) 0.75 μg/kgdose N 6 1 0 0 Mean 10.9 NQ NQ NQ Min-Max BLQ-18.8 18.6 NQ NQ 1.5 μg/kgdose N 6 2 1 4 Mean 20.9 NQ NQ 311 Min-Max 7.53-28.5 13.1-22.5 475268-359 2.25 μg/kg dose N 6 2 3 4 Mean 39.7 NQ 1561 678 Min-Max13.1-59.1 29.8-48.5 1551-1569 655-694 3.0 μg/kg dose, excluding subject1027 who had no quantification concentrations N 6 4 3 5 Mean 61.7 36.12257 965 Min-Max 53.9-76.0 27.7-51.3 1773-2764 823-1124

Platelet response to the administration of TPO Compound No. 1 wassimilar to published results for rhTPO and AMG531. Platelet countsincreased dose-dependently reaching peak levels at Day 10-12, and countsreturned to baseline within 3-4 weeks (FIG. 8). Mean peak plateletcounts ranged from 315×10⁹/L at 0.375 μg/kg i.v. to 685×10⁹/L at 3 μg/kgi.v., and mean maximal platelet counts increased from baseline rangedfrom 1.4-fold at 0.375 μg/kg to 3.2-fold at 3.0 μg/kg (Table 2). Atleast 50% increase in platelets was observed in 4 out 6 subjectsreceiving TPO Compound No. 1 at a dose of 0.75 ug/kg, while at least2-fold increase in platelet count was observed in about 3 out of 6subjects at a dose of 1.5 ug/kg, etc. The dose of 0.75 ug/kg i.v. hasbeen chosen as the starting dose for phase II clinical study.

Apart from changes in platelet counts, other mature circulating bloodcells were not affected (data not shown). In addition, administration ofTPO Compound No. 1 did not affect platelet function, not at the time ofadministration, nor at 12 days post-dose, at the time of the appearanceof newly produced platelets. TABLE 2 Summary of platelet count analysesTPO Compound 0 0.375 0.75 1.5 2.25 3.0 No. 1 (μg/kg) (μg/kg) (μg/kg)(μg/kg) (μg/kg) (μg/kg) N 10 6 6 6 6 6 n (>1.5×) 0 3 3 4 4 5 n (>2×) 0 01 3 4 5 n (>3×) 0 0 0 0 0 4 n (>4×) 0 0 0 0 0 1 Plt₀ 192/203 223/205212/212 228/230 215/203 208/200 (10⁹/L)¹⁾ (163-233) (159-304) (155-264)(200-258) (193-261) (150-284) Plt_(max) 230/225 315/309 347/335 430/458454/500 685/750 (10⁹/L)¹⁾ (189-271) (214-482) (232-495) (238-597)(254-576) (188-979) Plt_(max)/ 1.14/1.13 1.42/1.42 1.63/1.63 1.91/2.132.15/2.33 3.21/3.50 Plt₀ ¹⁾ (1.04-1.38) (1.08-1.81) (1.15-2.12)(1.01-2.59) (1.28-2.98) (1.25-4.52)

The effect of TPO Compound No. 1 administration on growth factors thatare known to possess thrombopoietic activity was assessed. EndogenousTPO levels dose-dependently increased, reaching peak levels at 3 dayspost-dose (FIG. 9). No significant changes were observed in blood levelsof IL-6, IL-11 and EPO levels.

Platelet function, assessed as collagen-induced platelet aggregation inwhole blood, was not different between the treatments. None of thesubjects experienced a serious adverse event or dose-limitingtoxicities. The most frequently observed adverse events included mildheadache and fatigue and occurred both after active treatment andplacebo. No antibodies against peg-TPOmp were detected. These resultsindicate that TPO Compound No. 1 was well tolerated at the tested doserange.

1. A compound that binds to a thrombopoietin receptor, wherein saidcompound comprises (H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH2, wherein X₁₀ isselected from the group consisting of sarcosine or β-alanine.
 2. Thecompound of claim 1, wherein said compound is covalently attached to ahydrophilic polymer.
 3. The compound of claim 2, wherein saidhydrophilic polymer has an average molecular weight of between about 500to about 40,000 daltons.
 4. The compound of claim 2, wherein saidhydrophilic polymer has an average molecular weight of between about5,000 to about 20,000 daltons.
 5. The compound of claim 2, wherein saidpolymer is selected from the group consisting of polyethylene glycol,polypropylene glycol, polylactic acid and polyglycolic acid.
 6. Thecompound of claim 5, wherein said compound is covalently attached topolyethylene glycol.
 7. The compound of claim 1, wherein each of thedimeric subunits of said compound is covalently attached to ahydrophilic polymer.
 8. A pharmaceutical composition comprising acompound of claim 1 in combination with a pharmaceutically acceptablecarrier.
 9. A method for treating a patient suffering from a disorderthat is susceptible to treatment with a thrombopoietin agonist,comprising administering to the patient a therapeutically effective doseor amount of a compound of claim
 1. 10. A physiologically active,substantially non-immunogenic water soluble polypeptide compositioncomprising a compound of claim 1 coupled with a coupling agent to atleast one polymer having a molecular weight of between about 500 toabout 20,000 daltons selected from the group consisting of polyethyleneglycol and polypropylene glycol, wherein said polymer is unsubstitutedor substituted by alkoxy or alkyl groups, said alkoxy or alkyl groupspossessing less than 5 carbon atoms.
 11. The polypeptide composition inaccordance with claim 10, wherein said polymer has a molecular weight ofabout 750 to about 15,000 daltons.
 12. The polypeptide composition inaccordance with claim 10, wherein said polymer has a molecular weight ofabout 5,000 to about 10,000 daltons.
 13. The polypeptide composition inaccordance with claim 10, wherein said polymer is polyethylene glycol.14. A substantially non-immunogenic water soluble polypeptidecomposition comprising a compound of claim 10 and a pharmaceuticallyacceptable carrier.
 15. A method of activating a thrombopoietin receptorin a cell, comprising contacting said cell with an effective amount of acompound which comprises (H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH2, wherein X₁₀is selected from the group consisting of sarcosine or β-alanine.
 16. Amethod according to claim 15 wherein said cells comprise humanmegakaryocytes, platelets or CD34+ cells.
 17. A method according toclaim 15 wherein said cells comprise TPO-dependent cells.
 18. A methodof treating thrombocytopenia in a subject, comprising: (a) obtaining apopulation of said subject's cells comprising megakaryocyte precursorcells; (b) treating said cells according to the method of claim 15; and(c) administering said treated cells to said subject, to increase thenumber of megakaryocytes present in said subject compared to that whichwould occur without such treatment.
 19. A method according to claim 18wherein said thrombocytopenia is due to chemotherapy.
 20. A methodaccording to claim 19 where said population of cells is obtained priorto said chemotherapy.
 21. A method according to claim 18 wherein saidthrombocytopenia is due to radiation therapy.
 22. A method according toclaim 21 where said population of cells is obtained prior to saidradiation therapy.
 23. A method of treating a patient suffering fromthrombocytopenia, comprising administering to said patient atherapeutically effective dose of a compound which comprises(H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH2, wherein X₁₀ is selected from the groupconsisting of sarcosine or β-alanine.
 24. A method according to claim 23wherein said thrombocytopenia is due to chemotherapy or radiationtherapy.
 25. A method according to claim 24 wherein a TPO antagonist isadministered to said patient prior to said chemotherapy or radiationtherapy.
 26. A method according to claim 23 wherein saidthrombocytopenia is due to bone marrow transfusion.
 27. A method ofprophylactically treating a patient at risk of thrombocytopenia,comprising administering to said patient a prophylactically effectiveamount of a compound which comprises (H-IEGPTLRQ(2-Nal)LAARX₁₀)₂K-NH2,wherein X₁₀ is selected from the group consisting of sarcosine orβ-alanine.
 28. A method according to claim 27 where said compound isadministered prior to bone marrow transplantation, chemotherapy, orradiation therapy.
 29. A compound that binds to thrombopoietin receptor,said compound having: (1) a molecular weight of less than about 8000daltons, and (2) a binding affinity to thrombopoietin receptor asexpressed by an IC₅₀ of no more than about 100 μM, wherein said compoundcomprises the following sequence of amino acids: X₉ X₈ G X₁ X₂ X₃ X₄ X₅X₆ X₇

where X₉ is A, C, E, G, I, L, M, P, R, Q, S, T, or V; X₈ is A, C, D, E,K, L, Q, R, S, T, or V, X₁ is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R,S, T or V; X₃ is C, F, I, L, M, R, S, V or W; X₄ is any of the 20genetically coded L-amino acids; X₅ is A, D, E, G, K, M, Q, R, S, T, Vor Y; X₇ is C, G, I, K, L, M, N, R or V, and X₆ isβ-(2-naphthyl)alanine.
 30. The compound of claim 29, wherein saidsequence of amino acids is cyclized.
 31. The compound of claim 29,wherein said sequence of amino acids is dimerized.
 32. 1. A method ofactivating a thrombopoietin receptor in a cell, comprising contactingsaid cell with an effective amount of a peptide having a molecularweight of less than about 8000 daltons, said compound comprises thefollowing sequence of amino acids: X₉ X₈ G X₁ X₂ X₃ X₄ X₅ X₆ X₇

where X₉ is A, C, E, G, I, L, M, P, R, Q, S, T, or V; X₈ is A, C, D, E,K, L, Q, R, S, T, or V, X₁ is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R,S, T or V; X₃ is C, F, I, L, M, R, S, V or W; X₄ is any of the 20genetically coded L-amino acids; X₅ is A, D, E, G, K, M, Q, R, S, T, Vor Y; X₇ is C, G, I, K, L, M, N, R or V, and X₆ isβ-(2-naphthyl)alanine.
 33. The compound of claim 32, wherein saidsequence of amino acids is cyclized.
 34. The compound of claim 32,wherein said sequence of amino acids is dimerized.
 35. A method ofactivating a thrombopoietin receptor in a cell, comprising contactingsaid cell with an effective amount of a compound covalently attached toa hydrophilic polymer, said compound comprises the amino acid sequence IE G P T L R Q (2-Nal) L A A R A.
 36. The method of claim 35 wherein saidhydrophilic polymer has an average molecular weight of between about 500to about 40,000 daltons.
 37. The method of claim 35 wherein saidhydrophilic polymer has an average molecular weight of between about5,000 to about 20,000 daltons.
 38. The method claim 35 wherein saidpolymer is selected from the group consisting of polyethylene glycol,polypropylene glycol, polylactic acid and polyglycolic acid.
 39. Themethod of claim 38 wherein said compound is covalently attached topolyethylene glycol.
 40. A method according to claim 35 wherein saidcells are in vivo.
 41. A method according to claim 35 wherein said cellsare in vitro.
 42. A method according to claim 35 wherein said cellscomprise human megakaryocytes, platelets or CD34+ cells.
 43. A methodaccording to claim 35 wherein said cells comprise TPO-dependent cells.44. A method of treating thrombocytopenia in a subject, comprising: (a)obtaining a population of said subject's cells comprising megakaryocyteprecursor cells; (b) treating said cells according to the method ofclaim 35; and (c) administering said treated cells to said subject, toincrease the number of megakaryocytes present in said subject comparedto that which would occur without such treatment.
 45. A method accordingto claim 44 wherein said thrombocytopenia is due to chemotherapy.
 46. Amethod according to claim 45 where said population of cells is obtainedprior to said chemotherapy.
 47. A method according to claim 44 whereinsaid thrombocytopenia is due to radiation therapy.
 48. A methodaccording to claim 47 where said population of cells is obtained priorto said radiation therapy.
 49. The compound of claim 29, wherein thecompound comprises the following amino acid sequence: I E G P T L R Q(2-Nal) L A A R A.